Although this functional emergence based on local environmental control is attracting a great deal of attention in chemistry, it remains challenging to realize this artificially. Herein, we report the tuning of the thermo-responsive properties of oligo(ethylene glycol) (OEG) derivatives attached on gold nanoparticles via local environmental control not only by the hydrophobic moiety at their terminus but also by their molecular configuration. OEG-attached alkane thiol-modified AuNPs showed thermo-responsive assembly/disassembly in water through the hydration/dehydration of the OEG portions in a manner dependent both on the hydrophobicity at their terminus and the surface curvature of the core nanoparticles. Further, the assembly temperature (TA) was also tuned by ligand mixing with a non-thermo-responsive ligand with a shorter OEG length. Molecular dynamics simulations show that the distribution of the hydrophobic terminus in the normal direction along the gold surface varied in accordance with the surface curvature, indicating variations in molecular configuration. It is expected that a bent configuration could accelerate the thermo-responsiveness of OEG by allowing them greater accessibility to the hydrophobic terminus. Experimental and simulation results support the notion that local OEG density tuning by surface curvature or ligand mixing with a different OEG length leads to different degrees of accessibility to the hydrophobic terminus via changes in molecular configuration, promoting local environmental control-directed assembly temperature tuning.Biomolecular systems actively control their local environment on a sub-nm scale  via local environmental control not only by the hydrophobic moiety at their terminus but also by their molecular configuration.Thermo-responsive properties of oligo(ethylene glycol) derivatives attached on gold nanoparticles were tuned  For example, in nature, peptides and proteins are produced by the connection of amino acids and show various properties which are not found in amino acids as monomers. Further, enzymes, which are proteins that take on sophisticated conformations 8–10 PEG and its derivatives are attached to vesicles and nanoparticles to improve their dispersibility and retentivity in blood. Further, OEG-attached alkane thiols are used for the surface modification of biosensors to prevent undesired adsorption.11,12 PEG possesses not only bio-compatibility and bio-stealth properties but also thermo-responsiveness on hydration/dehydration states, like a poly(N-isopropyl acrylamide) (pNIPAM).13,14 Despite their common features, pNIPAM but not PEG is widely used as a thermo-responsive polymer.15–17 This is probably due to the difficulties associated with the tuning of thermo-responsiveness to preferable temperatures.13,18 On the other hand, OEG-derivative-grafted polymers19,20 and OEG-derivative-attached detergent assemblies (micelles)21,22 also show thermo-responsiveness as hydration/dehydration phenomena. The copolymerization of two OEG macromonomers of different chain-lengths with different degrees of hydrophilicity leads to the formation of thermosensitive copolymers with a tunable lower critical solution temperature (LCST).23,24 These kinds of thermo-responsive polymers with tunable LCST are now under the spotlight due to their potential therapeutic applications.25 Yet despite the enormous potential, there are no reports to date on their function evolution via control over their molecular configuration or the local state of the OEG portion.Ethylene glycol-connected molecules, oligo and poly(ethylene glycol) (OEG and PEG), are particularly important synthetic molecules for medical and therapeutic applications due to their properties, such as neutral charge, water-solubility, and biocompatibility.26–28 However, when using AuNPs, surface modification remains an issue for the provision of stable dispersibility in solution. To this end, hydrophilic biocompatible polymers, such as PEG or OEG, are attached to their surfaces. Here, in terms of the surface modification of nanoparticles, there is one unmistakable fact: nanoparticle curvature controls the molecular conformation and chemical properties of the attached molecules by changing their local density or distances (in a range between 4–20 nm of AuNP diameter).29–31 Grzybowski et al. showed the unique possibilities for AuNP assembly regulated by electrostatics via location-specific charging on dumbbell-shaped nanoparticles as an effect of curvature.31 The shape of the core particle affords a way to control the molecular states within self-assembled monolayers and surface chemistries. This could be an advantage, not of polymers, but of small molecules or oligomers.32Recently, gold nanoparticles (AuNPs) have attracted much attention in terms of potential therapeutic applications due to their plasmonic properties.33 Importantly, their thermo-responsiveness shows a significant dependence on the core particle sizes. This size effect was confirmed as a surface curvature effect, not a volume effect, by using different sized rod-shaped gold nanoparticles.34 Further, we demonstrated the hierarchical self-assembly of rod-shaped gold nanoparticles based on curvature-dependent thermo-responsiveness.34 It seems that nanoparticle shape-based tuning of assembly temperatures (TA) is particularly useful in that it shows dramatic changes such as 67, 56, and 39 °C for gold nanoparticles of 3, 5, and 10 nm in diameter, respectively.33 However, as the exact control of NP size or curvature remains difficult, we could not precisely tune their TA, thus missing out on potential applications. In this study, we report a novel TA tuning approach for AuNPs coated with OEG-derivatives by local molecular environmental control of thermo-responsive OEG-derivatives (C2-EG6) via mixing with short ligands (HO-EG2), which are not thermo-responsive under the applied experimental conditions, as molecular configuration-directed tuning (We have previously reported that gold nanoparticles (AuNPs) covered with OEG-attached alkane thiols show thermo-responsive assembly phenomena caused by hydrophobic interactions from the dehydration of the OEG portions.d tuning .via ligand exchange reaction in water according the method described in our previous paper.33 Citric acid and excess ligands were removed, and the solvent was replaced with 10 mM HEPES buffer (pH 8.0) via repetitive centrifugal purifications. Results for dynamic light scattering (DLS), zeta-potential, and extinction spectral analyses support the successful surface modification  for the citrate-protected AuNPs to weak negative (−8.7 ± 4.9 and −10.6 ± 3.1 mV in buffer) for C1- and C2-EG6-coated AuNPs (d = 15 nm), respectively. Extinction spectra showed a slight red-shift of the plasmonic peak from 517, 520, and 532 nm for citrate-coated to 522, 523, and 532 nm for C2-EG6-coated of AuNPs , respectively, due to refractive index changes around the AuNPs.First, we prepared thermo-responsive AuNPs by surface modification with a C1- or C2-EG6 ligand. The AuNP  surfaces were modified with alkane thiol ligands 33 Importantly, DLS measurements showed clear changes in size distribution responding to temperature changes in the heating and cooling processes (TA) was next determined as 32.5 ± 1.0 °C from three independent DLS analyses on heating. In the same way, assembly temperatures (TA) of C2-EG6-coated AuNPs of different diameters (10 nm and 20 nm) were determined to be 43.5 ± 3.5 °C and 24.2 ± 1.5 °C, respectively  showed thermo-responsive assembly between 30 and 40 °C, which is similar to our previous reports .33 Imporrocesses . In the ectively . The TA termined . Fig. 1CTA with a 1 nm increase in diameter, suggesting that extreme control of AuNP size or shape can provide precise tunability. Although preparation techniques for nanoparticles have greatly advanced recently, size control on a sub-nm scale remains quite difficult.35 For example, the company (BBI Solutions) provides AuNPs of well-controlled size, which we used in this study, with a variation coefficient of within 8%. That is ca. 1 nm size distribution for 15 nm AuNPs, resulting in a 2 °C variation in TA. Thus, we performed assembly temperature tuning using ligand mixing as another approach using 15 nm AuNPs. First, we focused on the tuning of hydrophobicity by mixing surface ligands with different termini. For the surface modification of 15 nm AuNPs, we applied thermo-responsive ligands of C1-EG6 and C2-EG6 at various mixing ratios. These AuNPs showed obvious shifts in assembly temperature between 33 °C for 100% C2-EG6 and 63 °C for 100% C1-EG6 . In any case, it is possible to tune the assembly temperature by controlling the hydrophobicity at the terminus. The convex shape of these curves are similar to the clouding temperatures for binary mixture of various combinations of oligo(ethylene glycol)-based surfactants.36 In that report, the convex shapes were supported by Flory–Huggins theory extended to the binary surfactant system with an ideal mixing. Thus, these results are thought to be reasonable from the perspective of thermodynamics. Importantly, as the ligand mixing ratio is easily and precisely tunable, this is an effective approach.Precise tuning of the assembly temperature is an important issue for bio-related applications, particular tuning around 37 °C. % C1-EG6 . As it ipressive . Only 5%ca. 5 molecules nm−2 by inductively coupled plasma atomic emission spectroscopy (ICP-AES) and was independent of their size and shapes.33,34 Surface curvature induces a different local density of the OEG portion at the outer surface, although their molecular density at the AuNP surface remains the same. Thus, we hypothesized their local density, which is related to the molecular free volume, is one of the key factors in tuning their assembly temperature. To tune their local density, here, we mixed the thermo-responsive ligand, C2-EG6, with a non-thermo-responsive ligand with a shorter OEG chain-length, HO-EG2  on the control of their thermo-responsive assembly. In our previous paper, we showed that the surface ligand density on AuNPs was determined to be , HO-EG2 .TA by the ligand mixing ratios of C2-EG6 to HO-EG2 clearly indicate that the local molecular density tuned by mixing with a short-chain OEG ligand had a significant linear effect on TA  formation of alkane thiols on flat surfaces.37,38 These results indicate that significant differences in the hydrophobic tails is a key factor in the phase segregation in SAMs. Further, there are several reports showing domain formations in the SAM on AuNP surfaces using small particles, indicating that a smaller size in diameter, particularly one less than 5 nm, also has an effect.39,40 Although we could not determined the real ligand contents on the surface, these reports suggest that there is no significant difference between the ligand mixing ratios in solution and ligand content on the AuNP surface.In these experimental results, the C2-EG6 content on the TA shifts result from changes in the local molecular state in the OEG portion of the surface ligands on curved surfaces, molecular dynamics (MD) simulations were performed. Briefly, 63–65 ligand molecules were attached on a curved surface in a limited space to tune the ligand density to ca. 5 molecules nm−2  group at the terminus is trapped into the OEG layer more often on surfaces with a lower curvature via a bent conformation than that on surfaces with a higher curvature. That is, a higher local OEG density promotes and stabilizes access of the hydrophobic terminus to the OEG portions due to the relatively higher hydrophobic atmosphere together with changes in the molecular configuration. Thus, the radial distribution function of the terminal carbon atom against water molecules was also analyzed for each molecules around the central portion  values and particle sizes. Larger particles provided smaller solvation peak values, indicating that the carbon at the terminus is placed in a more hydrophobic environment. We also found that ligand molecules were sometimes trapped with an irregular configuration, in which the terminus was attached on the alkane thiols tails. These phenomena also support our notion described above. As this molecular bent configuration allows direct contact between hydrophobic terminus and the OEG portion, it can promotes their dehydration, causing a decrease in TA. That is, the molecular configuration derived from the local OEG density appears to be a critical factor. These MD simulation results suggested that different molecular configurations are provided by variations in the local OEG density due to different surface curvatures, thus inducing shifts in the thermo-responsivity of the OEG derivatives by differences in the degree of accessibility of the hydrophobic terminus.To confirm that these  portion . The shag(r) of the terminal carbon atom against the oxygen atom of the water molecule increased with decreases in C2-EG6 content (increases in HO-EG2 content)  Fig. S13. AlthougFig. S14B. These tFig. S14B and 3. Tvia the local environmental control not only by the hydrophobic moiety of their terminus but also by their molecular configuration. OEG-attached alkane thiol-modified AuNPs showed thermo-responsive assembly/disassembly through the hydration/dehydration of the OEG portions in a manner dependent on the hydrophobicity at the terminus. Further, the surface curvature of the core nanoparticles changes their responsive temperatures. Molecular dynamics simulations show that a hydrophobic terminus distribution along the normal direction to the gold surface varied in accordance with the surface curvature, indicating variations in the molecular configuration. A smaller curvature provides a higher local density, which promotes a bent configuration among the ligand molecules, probably due to the stabilization of the hydrophobic terminus in the relatively dense OEG moiety. This bent configuration could accelerate the thermo-responsiveness of the OEG portion via their greater degree of accessibility to the hydrophobic terminus. Importantly, the TA was also tuned by ligand mixing with a non-thermo-responsive ligand with a shorter OEG chain-length. Although the MD simulations do not directly support this experimental result, experimental and simulation results on the curvature dependence of TA support the notion that local OEG density tuning by surface curvature or ligand mixing with OEG of a shorter chain-length leads to differences in the degree of accessibility of hydrophobic terminus via changes in molecular configuration, thus promoting a local environment control-directed assembly temperature. As biomolecular systems actively control their local environment on a sub-nm scale via changes in their molecular configuration from their flexible structures and thereby derive emergent functions, our finding in this study provides a novel molecular design to the tuning of their functionality based on the molecular configuration control via local environmental tuning on a sub-nm scale as a biomimetic system, leading to opening a door for novel emergent functions.In this study, the thermo-responsive properties of oligo(ethylene glycol) (OEG) derivatives attached on gold nanoparticles were tuned 40,43 All commercially available reagents were used without further purification. All solvents were purchased from Wako Pure Chemical Industries Ltd. (Japan).Citrate-protected AuNPs in aqueous solution  were purchased from BBI Solutions (UK). Surface ligands with a methyl, ethyl, and hydroxyl head, referred to as C1-EG6, C2-EG6, HO-EG6, and HO-EG2, respectively, were synthesized according to our previous paper .40,43 Alvia a ligand exchange reaction. Briefly, 1 mL of citrate-coated AuNPs  was concentrated by centrifugation (12 000g for 45 min) and the following removal of the supernatant (900 μL). The concentrated citrate-protected 10 nm AuNPs , 15 nm AuNPs , and 20 nm AuNPs  were mixed with the thiolate ligand molecules including tris(2-carboxyethl)phosphine (TCEP) as a reductant. In this exchange reaction, the total ligand number was adjusted to 10 equivalent for the surface atoms on a AuNP.44 In the case of 10 nm AuNPs, the number of surface Au atoms was 4192/particle and the concentration of the ligand solution was 4.0 mM. In the case of 15 nm and 20 nm AuNPs, the number of surface Au atoms was 9585 and 17178/particle, respectively, and the ligand concentration for both was 2.0 mM. After the addition of ultrapure water up to 500 μL, the AuNPs were incubated for 48 h at 4 °C. Then, the ligand-exchanged AuNPs were washed 3 times by centrifugation (12 000g for 45 min), the removal of the supernatant (470 μL), and addition of 10 mM HEPES buffer (pH 8.0) up to 500 μL to remove the free thiol ligands.AuNPs coated with oligo(ethylene glycol) derivatives were prepared −1 followed by a waiting time of 2 min.The UV-vis-NIR spectra of the AuNPs were measured using a V-770 UV-Visible/NIR Spectrophotometer with PAC-743R Automatic 6 position Peltier cell changer . The temperature-change measurements of the AuNP spectra were performed at each temperature with changes at a rate of 1 °C minThe size and zeta-potential of AuNPs were measured using a Zetasizer Nano ZS . The temperature-change measurements for the AuNPs sizes were performed at each temperature after a waiting time of 2 min. The assembly temperature was defined as the middle temperature between the temperature at which the size of the AuNPs showed a significant change and the highest temperature at which the AuNPs remaining dispersed.R  simulations, partly due to the limitations in computing resources, we selected only a part of the particle surface for simulation in the absence of gold atoms. We employed this simplified model as the investigation of the local density dependence of OEGs on the curvature of the gold nanoparticle was our main computational purpose. For all the simulations, we used a cubic simulation box with a side length of 4 nm. The simulation boxes were set to intercept the top spherical cap of the nanosphere of gold nanoparticle with radius  Fig. S10.ca. 5 molecules nm−2. The spherical cap surface area formula was used to calculate the particle area in the box.According to the experimental condition, the ligand density on the particle surface was set at R is the radius of the sphere, and H is the height of the spherical cap, which can be easily obtained by the Pythagorean theorem.In the above formula, R = 10 nm, 15 nm, and 20 nm, respectively.From the calculated surface area, the number of ligands was 65, 64, and 63 for 245 was used in the modeling of the initial configuration of the ligand molecules. Furthermore, in order to make the oligomers evenly distributed on the surface of the spherical cap, the minimum distance between the molecules was set at 5 Å. An example of the configuration of C2-EG6 is shown in Fig. S11.†To randomly plant the thiol end of the ligands on the surface of the sphere cap, a Packmol programThe initial molecular structure and restrained electrostatic potential (RESP) charge of a single ligand were obtained by the AM1-bcc semi-empirical method using an antechamber program in Amber 14 ref.  tools. T−1 Å−2. This binding force to the sulfur atoms was also used for the following calculations. The elevation of temperature from 0 to 300 K and thermal equilibration calculation was similar to those for energy minimization, using isometric periodic boundary conditions and constraining sulfur atoms. In the equilibrium and MD calculations, The SHAKE method was used to constrain all the chemical bonds containing hydrogen atoms and ignore the chemical bond forces. After the system reached a target equilibrium temperature at 300 K, an equilibration calculation was performed for 100 ps using a Langevin thermostat. After equilibration, the main MD simulations were carried out for 20 ns at 300 K. As for the above equilibrium calculation, the SHAKE method and the Langevin constant temperature controller were used. Throughout all the calculation, the interatomic cut-off radius of coulombic force was set at 9.0 Å. To confirm the reproducibility of the simulation, the same calculations were performed twice.Energy minimization at 0 K was carried out under constant-volume periodic boundary conditions by the steepest descent method. During minimization, all the sulfur atoms of each oligomer were fixed on the sphere cap with a binding force of kcal molH. Mitomo designed the research and experiments. K. Xiong and Y. Shi performed experiments. X. Su and S. Sato performed MD simulation. The manuscript was written with contributions from all authors. All authors have given approval to the final version of the manuscript.There are no conflicts to declare.NA-003-D1NA00187F-s001NA-003-D1NA00187F-s002NA-003-D1NA00187F-s003NA-003-D1NA00187F-s004


The study assessed the level of self-care practice and its predictors among hypertensive patients in the health centers of Bole Sub-city, Addis Ababa, Ethiopia.A multi-Center-based cross-sectional study that employed 370 hypertensive participants at the conveniently selected Health Centers in Bole Sub-City; from August 01–30, 2020. The researchers selected the participants based on a simple random sampling method after applying for a pre-tested interviewer-administered questionnaire and secured for informed consent. All the statistical analyses were SPSS 22.0 software based. The authors used binary logistics regression to identify the presence and strength of association; with its respective 95%CI and p-value less than five percent as a significant level.The overall level of good self-care practice among hypertensive patients was 53.0% (95% CI: 47.2–58.8%) whereas 61.4%, 63.8%, 92.7%, 82.7%, and 18% of the study participants were adherent to medication, good weight management, non-smokers, alcohol abstainers and physical activity consecutively. Being illiterate had 2.347 and 2.084 times higher odds of having had good self-care practice compared to secondary school and a diploma or above consecutively. Being a merchant, civil, and retired were associated with good self-care practice than being unemployed.The study reported a lower level of self-care practice in the study settings. Educational level and occupation were factors identified for self-care practice. The authors recommended policymakers, healthcare workers, and researchers work on the identified factors of self-care practice of hypertensive participants in the study settings.The online version contains supplementary material available at 10.1186/s12872-023-03062-9. Hypertension; “the silent killer” is the top public health problem of both developed and the undeveloped world –6 affectBased on the recommendation by the World Health Organization (WHO), hypertensive patients should monitor their blood pressure (BP) after trained about the measurement procedure . Self-caWith the premise that the self-care practice by the hypertensive patient should encompasses maintenance of body weight, adherence to medication, waist circumference, physical exercise, dietary management and stress management, there was a global effort to reach at by the year 2025. These included reduction of the raised blood pressure by 25%, alcohol use and insufficient physical exercise by 10%, non-communicable disease by 25%, and population salt-intake and tobacco use by 30%. With all this efforts, different studies reported a lower level of self-care practice –18.An optimal self-care practice controls BP, and other cardiovascular disease related morbidities and mortalities . Though A multi-center-based cross-sectional study that received ethical approval from Santé Medical College, research review and ethics committee and Addis Ababa Regional research Review Committee and was conducted at the selected health centers in Addis Ababa, the capital of Ethiopia that has 10 sub-cities with 96 health centers. The researcher selected Bole sub-city, within Addis Ababa city, with a total of10 health centers providing healthcare service to about 415,572 population based on the report from the sub city. Proportionally, the sub city has 52% of female population. The researchers selected Bole sub-city conveniently, with the premise that healthcare service delivery system and procedure was similar across all the sub-cities within Addis Ababa. In Bole sub-city, there are ten health centers, five of which were included in the study based on simple random sampling method. The selected health centers were Bole Bulbula health center, Delfire health center and Amoraw Health Center /d2, with the assumptions of, 95% confidence interval (CI), 5% margin of error, and a self-care practice by hypertensive patient of 59.4% [The researchers carried out the study from August 01 to 30, 2022; ones secured a written informed consent from all the study participants following the approval from institutional review board (IRB) of Santé Medical College and research center of Addis Ababa city health bureau. The source population were all Hypertensive patients in Bole Sub-city, whereas the study population included 370 hypertensive patients, on follow-up and available during the data collection period at the selected Health centers fulfilling the inclusion criteria. Hypertensive patients should be on follow-up at least for six months in the selected health center, not critically by the time of data collection to respond to the questionnaire, and have no mental illness diagnosed clinically to be included in the study. Patient selection followed the principles of simple random sampling technique, ones obtained for their list from the respective health centers medical record. The sample size was determined based on population proportion estimation formula, n = zof 59.4% –25 then Outcome variable measurement: Self-care practice; the participants were regarded as having had good self-care practice if they scored above the median and poor if less than the median score of the constructs making up the self-care .In this particular study, self-care practice was measured by Hypertension self-care Activity level Scale effects (H-SCALE) that have six main domains; namely: medication adherence (3 items): Low-salt diet (12 items): scores of 6 out of 7 days-adherents. Physical activity (2 items): responses range from 0 to 14. Participants who scored 8 or better were considered adherent. Smoking (2 items): responses range from 0 to 14, and respondents who reported 0 days were considered a non-smoker, and all others were smokers. Weight management (10 items): responses range from 10 to 50. Participants who agreed or strongly agreed with all 10 items (score ≥ 40) were considered to have a good weight management practices. Alcohol (3 items): responses range from 0 to 21. Participants who did not take any alcohol in the last 7 days or who did not drink at all were abstainers.p-value of 0.136, R2 of 0.106, and Hosmer Lemeshow goodness of fit test- with a p-value of 0.775. Additionally, tested for multi collinearity tested with a Variance inflation factor for all the variables all showing a lower value than 0.05. Then after, selection of candidate variables done through bivariate logistics regression at a p-value of 0.20 in line with its respective 95% confidence interval and p-values. To control the effect of the confounding variable, a multivariable logistics regression was done, with its respective 95%CI and p-value less than 5% used as a level of significance.Data entry and cleaning were through Epi-Info version7, and then analyzed by statistical package for social sciences (SPSS) version 22.0 software after exported. Descriptive Statistics summarized the finding. Once the outcome variable dichotomized as poor and good self-care practice, factor identification was through binary logistics regression. After checked for model fitness, through testing for Omnibus test with a Totally 370 hypertensive patients approached from the Bole sub-city, and gave a response rate of 90%. The authors excluded all missed and incomplete data Fig. . The meaMost (60%) of the study participants had no family history of hypertension. More than one-third (36.5%) of the study participants had the hypertension with a duration of less than two years. In most (64.1%) of the study participants, type of hypertension was primary with the majority (84%) of the participants did not have a Sphygmomanometer at home Table .Table 2HAlmost all (96.2%) of the study participants took blood pressure pills, and 237 64.1%) of the participants took their medication at a constant time every day. Most (70%) of the study participants did not engage in a specific physical exercise . Educational status, age, gender, marital status, monthly income, duration since diagnosis and occupation of the study participants were candidate variables identified in the bivariate logistics regression analysis. In the multivariable logistics regression analysis, only educational status and occupation of the study participants were statistically associated with self-care practice.p < 0.05), and participants with a diploma and above  respectively.The odds of having had a good self-care practice was 2.347 and 2.084 times higher among illiterate participants compared to participants with secondary schooling , higher by 20% among Civil servants  and higher by 5% among retired  compared against unemployed participants  of the study participants had adhered to physical activity, which was lower than the finding of 57.3% in Saudi Arabia , and higIn this study, participants with a lower/no-educational level had good self-care practice. This was in line with the other different studies , 28. ThiLimitation of the study: This was a cross sectional study, establishing the temporal association was difficult on cause-effect relationship. The authors suggested a cohort study to identify factors having a direct risk on the self-care practice of hypertensive patients.This study reported a lower level of self-care practice by hypertensive patients. The educational level and occupation were factors identified having had statistically significant association with the self-care practice. Having a lower level of education, being a merchant or retired or civil servant was statistically associated with having had of lower self-care practice. The authors suggested policymakers, researchers, healthcare professionals to work on the identified problems in aggregation with every effort to prevent hypertension-related complications or promote the health of people with chronic medical conditions.Additional file 1. SI Hypertensive Patients Self-care activities in Health Centers of Addis Ababa, Ethiopia, November 2020 (n = 370).


Based on the obtained results, 5–15 nm SiNPs are able to induce the mitochondrial apoptosis pathway. Analyzed nanoparticles have also been found to cause an increase in selected oxidative stress parameters in both breast cancer cell lines. The presented study provides an explanation of the possible mechanisms of 5–15 nm SiNPs action in breast cancer cells.Breast cancer is one of the most common cancers in women. Silica nanoparticles (SiNPs) belong to the group of often-used nanoparticles in biomedical applications. The mechanisms of the cytotoxicity, apoptosis, and oxidative stress induced by the 5–15 nm SiNPs still remain unclear. The aim of the study was to evaluate the anti-cancer effect and mechanism of action of SiNPs in breast cancer cell lines. The breast cancer MDA-MB-231 and ZR-75-1 cell lines were analyzed using MTT assay, flow cytometry, and spectrophotometric methods. In this paper, we presented findings about the cytotoxicity, apoptosis, and oxidative stress in both breast cancer cell lines. We indicated that 5–15 nm SiNPs induced dose-dependent cytotoxicity in MDA-MB-231 and ZR-75-1 cells. Moreover, we demonstrated that the process of apoptosis in the studied cell lines was associated with a decrease in the mitochondrial membrane potential (ΔΨ The uncontrolled growth of breast cells leads to the development of breast cancer. This is one of the most common cancers in women, after lung cancer, with approximately 2 million new cases of breast cancer diagnosed in 2020. Factors that affect the formation of breast cancer are divided into two categories: modifiable and non-modifiable. Currently, most women who develop breast cancer are over the age of 50. Patient survival depends on the stage and molecular subtype of cancer. Based on mRNA gene expression levels, four basic molecular subtypes  can be distinguished among breast cancers, which allows for the implementation of new treatment strategies and patient stratifications that impact the management of breast cancer patients .A growing body of scientific evidence indicates that nanotechnology can meet the growing needs of current cancer therapies. Advanced biomaterials such as nanoparticles offer a unique opportunity to maximize the effectiveness of therapy while significantly reducing its toxic side effects . Silica Apoptosis is a complex, multi-stage process, the inhibition of which is the basis for the development of many cancers. Actions aimed at intensifying apoptosis are the basis for the development of new therapeutic strategies ,8,9.The process of apoptosis is inextricably linked with changes in the level of oxidative stress, especially with an increase in the level of reactive oxygen species (ROS). ROSs cause serious damage to cellular structures and macromolecules, such as proteins , lipids, and nucleic acids . The degThe reduced form of glutathione (GSH) is in close balance with the content of thiol groups in cellular proteins and protects cells from oxidative damage. One of the types of damage at the cellular level is the process of lipid peroxidation, which occurs mainly in the cytoplasmic membranes and is associated with lipid oxidation reactions. As a result of the above-mentioned chemical transformations, peroxide compounds and oxygen free radicals are formed, which activate and/or catalyze subsequent peroxidation reactions .There is a scarcity of data regarding the effect of SiNPs on the mechanism of apoptosis and oxidative stress conducted in breast cancer cells as a biological model. Moreover, the mechanism of action of SiNPs on breast cancer cells is still poorly understood. Therefore, we investigated the molecular mechanism of apoptosis and proved the influence of oxidative stress on the survival of MDA-MB-231 and ZR-75-1 cell lines. The inhibitory effect of 5–15 nm SiNPs on MDA-MB-231 and ZR-75-1 cells may be associated with increased cytotoxicity, ROS generation, TBARS content, decreased GSH/GSSG ratio, and the promotion of apoptosis. In our study, two different breast cancer cell lines, MDA-MB-231 and ZR-75-1, were used. These cell lines were chosen because of their specific morphology, the activity of genes related to apoptosis, the regulation of a cell cycle, and the expression of estrogen receptors . Data reThe apoptosis and necrosis in MDA-MB-231 and ZR-75-1 cell lines incubated with 5–15 nm SiNPs were designated using annexin V-FITC/PI double-staining by flow cytometry on FACSCanto II . The percentage of apoptotic and necrotic cells incubated in the medium in the presence of 100 μg/mL 5–15 nm SiNPs after 48 h is shown in After 48 h of incubation with 100 μg/mL 5–15 nm SiNPs, the percent of necrotic cells was similar to the control cells.m) in MDA-MB-231 and ZR-75-1 cells. After 48 h of incubation, a reduction in the mitochondrial membrane potential was demonstrated. The MDA-MB-231 and ZR-75-1 cells treated with 100 µg/mL 5–15 nm SiNPs showed an approximately 9-fold and 11-fold decrease in ΔΨm, respectively, in comparison to the untreated cells.Incubation with studied compounds caused changes in the TBARS content, which was analyzed as an index of lipid peroxidation D. StatisThe obtained results indicate that 5–15 nm SiNPs stimulate oxidative stress in breast cancer cells. The influence of 5–15 nm SiNPs and NAC on the amount of thiol groups is presented in The mechanisms of SiNPs cytotoxicity are not fully understood. The physicochemical properties of SiNPs are the binder in research on their cytotoxicity . The sizIn our current research, we used the MTT assay in order to test the cytotoxic effect of 5–15 nm SiNPs on MDA-MB-231 and ZR-75-1 breast cancer cells. The results obtained in the MTT assay showed that 5–15 nm SiNPs induced a decrease in the viability of MDA-MB-231 and ZR-75-1 cells in a dose-dependent manner. Ahamed et al. showed that SiNPs induce dose-dependent cytotoxicity in both A431 and A549 cells , and CuiAlthough the mechanism of 5–15 nm SiNPs cytotoxicity remains unclear, it may largely depend on their physicochemical properties, such as size, shape, structure, and elemental composition . In geneOxidative stress and ROS generation can be induced via SiNPs . Accordi2, is reduced to water. These reactions produce by-products, which include the superoxide anion (O−•), hydrogen peroxide (H2O2), hydroxyl radical (OH•), hydroperoxide radical (HO2•), and singlet oxygen (1O2). Depending on the concentration, ROS have the opposite effect in the cell. A moderate increase in their concentration may promote cell proliferation and survival, while exceeding a certain threshold level of ROS may lead to disturbances in the functioning of antioxidant systems, resulting in cell death [Oxidative stress is defined as an imbalance between the formation of free radicals and the abilities of individual defense mechanisms. The production of free radicals is a constant element of the cell’s oxygen metabolism. It is well known that oxidative stress causes many pathological conditions and diseases. Oxidative stress causes damage to proteins and lipids, as well as mutations and epigenetic disorders, thus leading to cancer or neurodegenerative diseases. ROS are atoms or molecules that have one unpaired electron on the valence shell. It is a broader concept than just free oxygen radicals as it also includes singlet oxygen and hydrogen peroxide. Under physiological conditions, the main source of ROS is the mitochondrial electron transport chain, in which molecular oxygen, Oll death . Many stll death ,21. In vll death . Cui et ll death . Guo et ll death . Petrachll death . We suggMany studies showed that apoptosis and necrosis are the main types of cell death ,7,10. PrSeveral studies indicated that SiNPs can be transported into the cell by diffusion, while larger SiNPs either settle on the cell surface or are absorbed by endocytosis. Smaller SiNPs can penetrate the cell interior and induce oxidative damage to the mitochondrial membrane, while the largest SiNPs tend to induce membrane injury by mechanical friction or oxidative damage .Numerous pro-apoptotic factors are released into the cytosol through the permeabilization of the outer mitochondrial membrane. These factors promote caspase activation. Next, cytochrome c binds to apoptotic protease activating factor-1 (Apaf-1) and leads to the assembly of the apoptosome complex .m, which was observed especially in ZR-75-1 cells. Previously, we presented that 5–15 nm SiNPs induced the loss of ΔΨm and changes in the mitochondrial ultrastructure, and we also proved that 5–15 nm SiNPs activated apoptosis in LBC3 glioma cells via the mitochondrial pathway [m inducing ROS production and apoptosis [In the activation of an intrinsic pathway of apoptosis in human/mammalian cells, an important role is played by mitochondria . We obta pathway . Accordipoptosis . TherefoIn summary, in the conducted research, we find a similar mechanism of action of SiNPs on MDA-MB-231 and ZR-75-1 cells in comparison with Ahamed et al.’s study on human skin epithelial A431 and human lung epithelial A549 cell lines. They investigated the effect of SiNPs in the concentration range of 25 to 200 μg/mL . In our Our research showed that 5–15 nm SiNPs activated caspase-9 and caspase-3. Subsequently, the apoptosome binds to caspase-9, and it goes on to activate caspase-3, which appears to be an event common in SiNPs-induced apoptosis. Ahamed et al. showed that silica nanoparticles increased caspase-3 and caspase-9 activity in A431 and A549 cells . ConsistTM-I, trypsin-EDTA, RPMI Medium 1640 (1×) + GlutaMAXTM-I, DPBS, penicillin, streptomycin, and fetal bovine serum Gold (FBS Gold) were provided by Gibco .Media for cells cultivation: L-15 Medium (1×) + GlutaMAXTM ; FAM-FLICA Caspase-9 Assay was provided by ImmunoChemistry Technologies . Chemical treatment of cells: silica nanoparticles 5–15 nm (SiNPs) were obtained from Sigma-Aldrich .Agents for apoptosis detection: JC-1 MitoScreen Kit, PE Active Caspase-3 Apoptosis Kit, and FITC Annexin V apoptosis detection Kit I were obtained from BD Pharmingen2DCFDA) were provided by Sigma-Aldrich  and GSH/GSSG-GloTM Assay kit by Promega .Chemical compounds for oxidative stress analysis: SDS, TCA, TBA, Folin-Ciocalteu reagent, N-acetylcysteine, and 2’,7’-dichlorodihydrofluorescein diacetate . Cells were cultured as described in our previous paper .To avoid the problem with the aggregation of 5–15 nm SiNPs, 5–15 nm SiNPs were dispersed in deionized water (1 mg/mL) by a sonicator, Sonopuls , on ice for 45 min . During experiments, cell culture media were replaced with a new one containing 5–15 nm SiNPs suspensions, at concentrations ranging from 12.5 μg/mL to 1000 μg/mL. Negative control constituted non-treated cells. For the analysis of cell viability, the 5–15 nm SiNPs concentration range was from 12.5 to 1000 µg/mL. In a separate MTT and oxidative stress experiment, cells were pre-treated (for 1 h) with NAC (5 mmol/L). Subsequently, cells were exposed to 5–15 nm SiNPs for 48 h. Apoptosis and necrosis detection and mitochondrial membrane potential (∆Ψm) analysis were conducted under the influence of 5–15 nm SiNPs at 100 μg/mL concentration. For the analysis of caspase-9 enzymatic activity, cells were treated with 100 µg/mL 5–15 nm SiNPs for 48 h. In the caspase-3 assay, cells were treated with 100 µg/mL of 5–15 nm SiNPs for 48 h.Cell viability was measured based on the methods of Carmichael et al. using 3--2,5-diphenyltetrazolium bromide (MTT) as described in our previous studies .Detection of apoptosis and necrosis was conducted with the use of FACSCanto II flow cytometry  as described in our previous studies .Disruption of the mitochondrial membrane potential was analyzed with the use of the MitoScreen kit , according to the manufacturer’s guidelines as it was described previously .Caspase-9 activity was measured using the FAM-FLICA Caspase 9 Kit (ImmunoChemistry Technologies) according to the manufacturer’s instructions as described in our previous studies .Caspase-3 was measured using the PE Active Caspase-3 Apoptosis Kit according to the manufacturer’s instructions as described in our previous studies .The level of intracellular ROS was determined using a dichlorodihydrofluorescein diacetate (DCFH-DA) assay,  as described in our previous studies .5 cells/mL) were incubated in 2 mL of medium with or without the test 5–15 nm SiNPs (100 μg/mL) in 6-well plates.SH groups were measured using the method of Rice-Evans , as desc5 cells/mL) were incubated in 2 mL of medium with or without the test 5–15 nm SiNPs (100 μg/mL) in 6-well plates.The level of membrane lipid-peroxidation products, or TBARS, was measured using the method of Rice-Evans , as descThe total glutathione content and GSH/GSSG ratio were studied with the use of a GSH/GSSG-Glo™ kit  following the manufacturer’s instructions. Cells of both cell lines were cultured in white 96-well plates and exposed to 5–15 nm SiNPs (100 μg/mL).Prior to the assay, growth media were removed, and cells were washed with PBS. The assay is based on a luminescence measurement and detects and quantifies total glutathione (GSH +GSSG), GSSG, and GSH/GSSG ratios in cultured cells. Stable luminescent signals are correlated with either the GSH or GSSG concentration of a sample. In this method, the GSH-dependent conversion of a GSH probe, Luciferin-NT, to luciferin by a glutathione S-transferase enzyme is coupled to a firefly luciferase reaction. The light from luciferase depends on the amount of luciferin formed, which in turn depends on the amount of GSH present. Thus, the luminescent signal is proportional to the amount of GSH. GSH/GSSG ratios are calculated directly from the luminescence measurements.5 cells/mL) were incubated in 2 mL of medium with or without the test compound in 6-well plates. The cells were then homogenized at 4 °C, and the total protein concentration in 0.1 M NaOH extract was calculated. Protein concentration was determined spectrophotometrically according to the method of Lowry et al [The MDA-MB-231 and ZR-75-1 cells  were calculated. The data were statistically analyzed using one way-ANOVA followed by Tukey’s post hoc m and an increase in the activity of caspase-9 and caspase-3. Obtained results indicate that 5–15 nm SiNPs are able to induce the mitochondrial pathway of apoptosis. However, 5–15 nm SiNPs have also been found to cause an increase in the oxidative stress level in MDA-MB-231 and ZR-75-1 cells (In summary, the present study indicated that 5–15 nm SiNPs induced cytotoxicity and apoptosis in both studied cell lines. The level of apoptosis in MDA-MB-231 and ZR-75-1 cells was correlated with an observed decrease in ΔΨ-1 cells . The preTaken together, our results may contribute to a better understanding of the coexistence of two phenomena, apoptosis and oxidative stress, and their possible role in breast cancer treatment. The novelty of the presented study consists mainly in analyzing the possible mechanisms of 5–15 nm SiNPs cytotoxic action and their role in the induction of apoptosis. Therefore, we suggest that 5–15 nm SiNPs may be a useful tool in the treatment of breast cancer.


Dry eye disease (DED) is a common and growing eye problem worldwide. Chronic DED symptoms can, subsequently, affect the patients’ quality of life (QOL). This prospective cross-sectional study aimed to assess the reliability, validity, and responsiveness of the Thai version of the Dry Eye-Related Quality-of-Life Score (DEQS-Th) questionnaire and to evaluate its accuracy in DED screening. Psychometric validation was conducted on DED participants. All participants completed the DEQS-Th and other measurements including the Ocular Surface Disease Index (OSDI) and the 5-level EQ-5D (EQ-5D-5L). Internal consistency, concurrent validity, convergent, and discriminant validity were evaluated. The standardized response mean (SRM) was used to evaluate the responsiveness of the DEQS-Th. The optimal cut-off score of DEQS-Th for DED screening was assessed. Among 100 participants with a mean age of 50.9 ± 14.4 years, and 89.0% female, the internal consistency of the DEQS-Th was excellent . The test-retest intraclass correlation was 0.82–0.92. It showed concurrent validity with the OSDI  and EQ-5D-5L index scores . DED is suspected if the DEQS-Th score ≥ 18.33  or its Short Form score ≥ 3 . The SRM of the symptom subscale of DEQS-Th was 0.82, indicating relatively large responsiveness, whereas the impact on daily life subscale and the summary score was small. In conclusion, the DEQS-Th is valid and reliable for evaluating the multifaceted effects of DED on a patient’s QOL. It can be useful for primary assessment and monitoring of DED in routine clinical practice. Dry eye disease (DED) is characterized by a vicious cycle of tear film instability and hyperosmolarity leads to ocular surface inflammation and damage, and neurosensory abnormalities . It is aAccording to the International Dry Eye Workshop 2017(DEWS II) report, the diagnosis of DED involves the presence of symptoms assessed by a dry eye questionnaire as well as any of three specific signs including tear break-up time (TBUT), tear osmolarity, and ocular surface staining . LikewisEven though symptoms and signs of dry eye are inconsistent and vary across individuals and DED sub-types ,12, the Currently, several dry eye questionnaires have been used in gathering symptoms in clinical research and practice . HoweverDespite the high prevalence of DED in Thailand , limitedThis prospective cross-sectional study was approved by the Institute Review Board before being initiated (study code: OPT-2561-005562) and followed the Declaration of Helsinki. All volunteers signed a written informed consent after a complete explanation.One hundred participants, diagnosed as DED at the Ophthalmology Clinic, Chiang Mai University Hospital, were recruited between 2018–2019. The eligible criteria included adult subjects aged ≥ 18, voluntary participation, and literate in Thai. The criteria for diagnosis of DED complied with those defined by the DEWS II. All participants underwent complete ophthalmic examination for both eyes including measuring of visual acuity, intraocular pressure, and additional dry eye tests such as corneal fluorescein staining (CFS), TBUT, and basic tear secretion (Schirmer’s test). CFS scores were assigned based on a modified van Bijsterveld grading system  . TBUT waAll DED participants were asked to complete the DEQS-Th questionnaire and additional health-related QOL questionnaires including the OSDI and the 5-level EuroQol‑5‑Dimensions (EQ-5D-5L).To evaluate the reproducibility, the DED participants completed the DEQS-TH twice. The re-test was performed two weeks after the first test.To evaluate the responsiveness or change of the questionnaire regarding the response to treatment, ten patients received diquafosol tetrasodium 3% ophthalmic solution six times/day for treatment of DED. This eye drop is a purinergic P2Y2 receptor agonist on the ocular surface. It stimulates both water and mucin secretion from conjunctival epithelial cells and goblet cells, thereby rehydrating the ocular surface independent of tear secretion from the lacrimal glands –23. PartThe DEQS questionnaire contains 15 questions divided into two subscales: Bothersome Ocular Symptoms (6 questions) and Impact on Daily Life (9 questions). Each question has columns A and B for the frequency and severity, respectively. Response to the frequency portion in column A is based on a 5-point scale ranging from “none of the time” (0) to “all of the time” (4). A frequency score of 1–4 points prompts the respondent to proceed to the severity in column B to answer regarding the degree of severity on a four-point scale. The DEQS score is calculated with the following formula:  x 25/ . The score “0” indicates the best possible score (no symptoms) and “100” indicates the worst possible score (maximum symptoms) .The DEQS-Th was developed from the English version of the DEQS questionnaire . In brieThe OSDI consists of 12 questions with 3 subscales: ocular symptoms, vision-related function, and environmental triggers. Each DED patient rated symptoms on a 5-point scale from 0 (never) to 4 . The OSDI total score was obtained by multiplying the sum scores of all questions answered by 25 and dividing by the total number of questions answered, giving the OSDI a scale from 0–100 with higher scores reflecting greater disability. According to the OSDI scores, the patients were classified as normal (scores 0–12), mild (13–22), moderate (23–32), and severe (33–100) symptoms Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly**********   2. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: Yes**********   3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: No**********   4. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: Yes**********   5. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: ReviewThe manuscript reports on a follow-up investigation of the author’s previous article on the Dry Eye-related Quality-of-life Score (DEQS) questionnaire translated to the Thai language, DEQS-Th, for use in Thailand. They have used statistical methods for producing quantitative measures of the effectiveness of the questionnaire for screening patients with suspected dry eye disease (DED). They compared DEQS-Th with other DED tests and evaluated reliability, internal consistency, test-retest correlation, responsiveness, and accuracy of the DEQS-Th.The authors report cut-off scores to be used for screening and detecting DED using the DEQS-Th. Inconsistencies between the Abstract, Results and Discussion are noted below. However, there is a fundamental issue relative to the cut-off scores that should be addressed. The DEQS-Th cut-off scores are reported as “>X” for diagnostic tests and “>Y” for ocular symptoms. As can be seen from the Supplemental Tables S1 and S2, as the score rises, the sensitivity of the test decreases while the specificity increases. This is a standard property of Receiver Operator Characteristic (ROC) curves . Is it preferable to have a higher cut-off with decreased sensitivity (fewer cases of DED detected) and increased specificity ? Or is it better to have a lower cut-off with increased sensitivity (more cases of DED detected) and decreased specificity ? The authors should acknowledge that using a cut-off value represents a trade-off between sensitivity and specificity. If they recommend using “>” with the cut-off, they should justify the choice being made between sensitivity and specificity.Some specific comments:Lines 30-31, 281-282, 344: The cut-off scores, “>18.33“ and “>19”, for rating the DEQ-Th diagnostic tests are not equivalent. As can be seen in the Supplementary S1 Table, there is a difference between a cut-off of 18.33 and any other cut-off value. Only 18.33 gives the sensitivity of 90 and specificity of 76.67. For any other cut-off number, the sensitivity and specificity will be different. To report a cut-off of 19, the authors should recalculate the sensitivity and specificity corresponding to the cut-off and report those results in lines 281-282. In any event, the reported cut-off, sensitivity and specificity in the Abstract (lines 30-31) and the cut-off in the Discussion (line 344) need to be changed to be consistent with the results reported in the Results on lines 281-282. In addition, “>” is inappropriate if the reported cut-off is supposed to represent a particular sensitivity and specificity.Lines 31-32, 283-284, 350: The cut-off scores, “>3” and “>4”, for rating the DEQ-Th using subscale ocular symptoms for DED screening are not equal. As can be seen in the Supplementary S2 Table, there is a difference between a cut-off of 3 and a cut-off of 4. A cut-off of 3 gives the sensitivity equal to 93.00 and specificity equal to 63.33, while a cut-off of 4 gives a sensitivity of 86.00 and a specificity of 66.67. The cut-off, sensitivity and specificity given in the Abstract (lines 31-32) and the cut-off in the Discussion (line 350) need to be consistent with the cut-off, sensitivity and specificity given in the Results in lines 283-284. As noted above, here too, “>” is inappropriate if the reported cut-off is supposed to represent a particular sensitivity and specificity.Lines 73: The DEQS was developed in 2013, not 2014.Lines 148-154: For the OSDI, the patients use a 5-point scale, 0-4. Need to specify how this is translated to a 0-100 scale – to obtain the total OSDI score, calculate the sum of scores of all questions answered times 25 divided by total number of questions answered, giving the OSDI a scale from 0-100.Lines 209, 220, 226: none-DED should be non-DEDLines 421-423: Add the date of the FDA document, December 2009, to Ref 17 and update the availability date from 2021 to be current in 2022.Supplementary Material: The results presented in this manuscript, Tables 2-7 and S1 and S2, are based on data collected in the course of this research. The data have not been made available with this submission.**********   6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Reviewer #1: Nohttps://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.
 While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  24 May 2022Response to ReviewersDear Reviewer,We greatly appreciate your time for your kind review and constructive comments on the manuscript entitled “Reliability, validity, and responsiveness of the Thai version of the Dry Eye-Related Quality-of-Life Score questionnaire” (Submission ID PONE-D-22-00892). We have carefully studied your comments and made corrections as suggested. Please find enclosed the revised version of our manuscript highlighted in yellow and the detailed point-by-point response to the comments raised by the reviewer.We hope that our responses would be satisfactory.Sincerely yours,Napaporn Tananuvat, M.D.Reviewer #1: ReviewThe manuscript reports on a follow-up investigation of the author’s previous article on the Dry Eye-related Quality-of-life Score (DEQS) questionnaire translated to the Thai language, DEQS-Th, for use in Thailand. They have used statistical methods for producing quantitative measures of the effectiveness of the questionnaire for screening patients with suspected dry eye disease (DED). They compared DEQS-Th with other DED tests and evaluated reliability, internal consistency, test-retest correlation, responsiveness, and accuracy of the DEQS-Th.The authors report cut-off scores to be used for screening and detecting DED using the DEQS-Th. Inconsistencies between the Abstract, Results and Discussion are noted below. However, there is a fundamental issue relative to the cut-off scores that should be addressed. The DEQS-Th cut-off scores are reported as “>X” for diagnostic tests and “>Y” for ocular symptoms. As can be seen from the Supplemental Tables S1 and S2, as the score rises, the sensitivity of the test decreases while the specificity increases. This is a standard property of Receiver Operator Characteristic (ROC) curves . Is it preferable to have a higher cut-off with decreased sensitivity (fewer cases of DED detected) and increased specificity ? Or is it better to have a lower cut-off with increased sensitivity (more cases of DED detected) and decreased specificity ? The authors should acknowledge that using a cut-off value represents a trade-off between sensitivity and specificity. If they recommend using “>” with the cut-off, they should justify the choice being made between sensitivity and specificity.Response: Thank you for your comments and suggestions. We apologize for the inconsistencies. The data are now corrected. We strongly agree with you that there is a trade-off between sensitivity and specificity using the different cut-off scores. That’s why we use a cut-off of ≥18.33 that yields a sensitivity of 90.00% and a specificity of 76.67%. This optimal criterion also provides the lowest cost (0.131) when the cost estimation is taken into account. In the revised manuscript, we apply “≥” for the criterion values in both Supplement S1 Table and S2 Table as suggested.Some specific comments:Lines 30-31, 281-282, 344: The cut-off scores, “>18.33“ and “>19”, for rating the DEQ-Th diagnostic tests are not equivalent. As can be seen in the Supplementary S1 Table, there is a difference between a cut-off of 18.33 and any other cut-off value. Only 18.33 gives the sensitivity of 90 and specificity of 76.67. For any other cut-off number, the sensitivity and specificity will be different. To report a cut-off of 19, the authors should recalculate the sensitivity and specificity corresponding to the cut-off and report those results in lines 281-282. In any event, the reported cut-off, sensitivity and specificity in the Abstract (lines 30-31) and the cut-off in the Discussion (line 344) need to be changed to be consistent with the results reported in the Results on lines 281-282. In addition, “>” is inappropriate if the reported cut-off is supposed to represent a particular sensitivity and specificity.Response: Thank you for your careful review. In the revised manuscript, the cut-off score of ≥ 18.33 was used consistently in the Abstract, the Results, and the Discussion part. Practically, we suggest a cut-off score of ≥ 18 for simple use of the DEQS-TH questionnaire even though the exact sensitivity and specificity may be slightly changed from the original 18.33.This statement is added in the Discussion part (lines 346-347).Lines 31-32, 283-284, 350: The cut-off scores, “>3” and “>4”, for rating the DEQ-Th using subscale ocular symptoms for DED screening are not equal. As can be seen in the Supplementary S2 Table, there is a difference between a cut-off of 3 and a cut-off of 4. A cut-off of 3 gives the sensitivity equal to 93.00 and specificity equal to 63.33, while a cut-off of 4 gives a sensitivity of 86.00 and a specificity of 66.67. The cut-off, sensitivity and specificity given in the Abstract (lines 31-32) and the cut-off in the Discussion (line 350) need to be consistent with the cut-off, sensitivity and specificity given in the Results in lines 283-284. As noted above, here too, “>” is inappropriate if the reported cut-off is supposed to represent a particular sensitivity and specificity.Response: Thanks again for your comment. The cut-off score ≥ 3 was used consistently in the revised manuscript. Lines 73: The DEQS was developed in 2013, not 2014.Response: Thank you for the correction. The year of the DEQS development is changed to be… “2013” (line 73).Lines 148-154: For the OSDI, the patients use a 5-point scale, 0-4. Need to specify how this is translated to a 0-100 scale – to obtain the total OSDI score, calculate the sum of scores of all questions answered times 25 divided by total number of questions answered, giving the OSDI a scale from 0-100.Response: The calculation of the OSDI total score is added up as suggested as… “The OSDI total score was obtained by multiplying the sum scores of all questions answered by 25 and dividing by the total number of questions answered, giving the OSDI a scale from 0-100 with higher scores reflecting greater disability” (Lines 150-152).Lines 209, 220, 226: none-DED should be non-DEDResponse: Thanks for your careful review. These typing errors are corrected. We also re-check thorough the manuscript for any other mistakes.Lines 421-423: Add the date of the FDA document, December 2009, to Ref 17 and update the availability date from 2021 to be current in 2022.Response: Thank you for the suggestion. This reference is edited as suggested (line 425).Supplementary Material: The results presented in this manuscript, Tables 2-7 and S1 and S2, are based on data collected in the course of this research. The data have not been made available with this submission.Response: We believe that all relevant data are within the paper. However, the data are enclosed with this revision.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 27 Jun 2022Reliability, validity, and responsiveness of the Thai version of the Dry Eye-Related Quality-of-Life Score questionnairePONE-D-22-00892R1Dear Dr. Tananuvat,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Adrienne Csutak, MD, PhD, MScAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 11 Jul 2022PONE-D-22-00892R1 Reliability, validity, and responsiveness of the Thai version of the Dry Eye-Related Quality-of-Life Score questionnaire Dear Dr. Tananuvat:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Adrienne Csutak Academic EditorPLOS ONE


Chemerin is an extracellular protein with chemotactic activities and its expression is increased in various diseases such as metabolic syndrome and inflammatory conditions. Its role in lung pathology has not yet been extensively studied but both known pro- and anti-inflammatory properties have been observed. The aim of our study was to evaluate the involvement of the chemerin/ChemR23 system in the physiopathology of COVID-19 with a particular focus on its prognostic value.Blood samples from confirmed COVID-19 patients were collected at day 1, 5 and 14 from admission to Erasme Hospital (Brussels – Belgium). Chemerin concentrations and inflammatory biomarkers were analyzed in the plasma. Blood cells subtypes and their expression of ChemR23 were determined by flow cytometry. The expression of chemerin and ChemR23 was evaluated on lung tissue from autopsied COVID-19 patients by immunohistochemistry (IHC).21 healthy controls (HC) and 88 COVID-19 patients, including 40 in intensive care unit (ICU) were included. Plasma chemerin concentration were significantly higher in ICU patients than in HC at all time-points analyzed (p<0.0001). Moreover, they were higher in deceased patients compared to survivors (p<0.05). Logistic univariate regression and multivariate analysis demonstrated that chemerin level at day 14 of admission was an independent risk factor for death. Accordingly, chemerin levels correlated with inflammatory biomarkers such as C-reactive protein and tumor necrosis factor α. Finally, IHC analysis revealed a strong expression of ChemR23 on smooth muscle cells and chemerin on myofibroblasts in advanced acute respiratory distress syndrome (ARDS).Increased plasma chemerin levels are a marker of severity and may predict death of COVID-19 patients. However, multicentric studies are needed, before chemerin can be considered as a biomarker of severity and death used in daily clinical practice. Further studies are also necessary to identify the precise mechanisms of the chemerin/ChemR23 system in ARDS secondary to viral pneumonia. Chemerin is an extracellular protein first described in the early 2000’s . It is sCirculating chemerin levels are increased in various pathologies notably in metabolic syndrome, inflammatory conditions such as rheumatoid arthritis, inflammatory bowel diseases and cancers –13. The The COVID-19 pandemic is caused by a coronavirus named SARS-CoV-2 . CoronavGiven that chemerin is elevated in most of the pathologies associated with a higher risk of developing severe COVID-19 and seems to be associated with lung inflammation, in the present study, we evaluated the chemerin/ChemR23 system in COVID-19 patients and correlated it with disease severity.This prospective observational study was conducted on adult patients (from 18 to 70 years) with a confirmed COVID-19 infection (positive polymerase chain reaction (PCR) test performed on a nasopharyngeal swab), admitted to Erasme Hospital . These patients were further classified into three groups: non-hospitalized (NH), hospitalized in a conventional care unit (H) and hospitalized in intensive care unit (ICU). The study was approved by the local Ethical Committee .Blood samples from confirmed COVID-19 patients were collected from April to December 2020 and stored at the Biobank BB190012 from the Laboratory of Vaccinology and Immunology at the Université libre de Bruxelles (ULB), Brussels, Belgium. Three different time points were analyzed: day 1 (D1), day 5 (D5) and day 14 (D14) from admission. As controls, blood samples from health care workers participating to a seroprevalence study  in anothClinical data were obtained from patient’s medical records with a focus on the following characteristics: age, gender, body mass index (BMI), smoking, comorbidities associated with a higher risk to develop a severe COVID-19 , chronic kidney disease and immunosuppression), peripheral oxygen saturation (SpO2) at admission, the need of ventilation, the development of an ARDS, the duration of hospitalization and outcome. Of note, ARDS was defined according to Berlin criteria as the development of breathing difficulties with hypoxemia  <300 mmHg) associated with bilateral opacities on chest imaging in the absence of cardiac failure . The res® aScopeTM, Ballerup, Denmark) as described by Taton et al.  collection. In this cohort, BALs were performed to confirm a SARS-CoV-2 infection or exclude opportunistic superinfections (immunocompromised patients). BAL were collected from April 2020 to November 2020. They were performed, using a disposable video-bronchoscope .Plasma and BAL chemerin levels were assessed by enzyme-linked immunosorbent assay (ELISA) . Samples were analyzed in duplicates according to the manufacturer’s instructions. Plasma levels of the Krebs Von den Lungen protein (KL-6) were detected using a sandwich ELISA assay on Lumipulse G600II and G1200 . The concentrations of IFN-α, IFN-γ, IL-1β, IL-6, IL-7, IL-8, IL-10, IL-17α, monocyte chemoattractant protein-1 , monokine induced by interferon-γ , macrophage inflammatory protein-1β  and tumor necrosis factor α (TNFα) were measured in plasma by multiparameter-based immunoassays  according to the manufacturer’s instructions. Results were analyzed with a Bio-Plex 200Whole blood cells conserved into the Cytodelics Stabilizer were prepared for flow cytometry analysis according to the manufacturer’s instructions. After complete lysis of red blood cells, the immune cells were stained with antibodies from Thermofisher : CD45 (EF450), CD3 (EF506), CD123 (SB600), CD11c (SB645), HLA-DR (SB702), CD16 (SB780), CD14 (FITC), CD141 (PE-Cy7), CD56 (APC-EF780) and ChemR23 (APC). Flow cytometry analysis were performed on an LSR Fortessa instrument  and analyzed using FlowJo software  to identify different immune cell populations and their expression of ChemR23. The gating strategy was previously described by Albayrak et al.  lung tissues from PCR positive COVID-19 patients stored in the Biobank ULB-COVID-19  were used for immunohistochemical (IHC) staining. Autopsies were performed after 72 hours to prevent infection of the medical staff . For thiIHC staining was performed using an automated immunostainer . Thus, 4µm slides were incubated respectively with anti-ChemR23  and anti-chemerin  antibodies and counterstained with hematoxylin. Then slides were scanned with a 40x magnification  before assessment by two pathologists.post-hoc tests. When only two groups were compared, student t or Mann-Whitney tests were used. Data are reported as mean ± standard deviation (SD) or median with a confidence interval of 95% (CI 95%). Categorical variables are listed as numbers with percentage. Chi-square test was performed to compare more than two groups and Fisher’s exact test was used for comparison between 2 groups. For correlation analysis, non-parametric Spearman correlation was performed, and data are reported as Spearman r and p-value. To determine the association of different variables to fatal outcome, a univariate logistic regression test was performed and then a multivariable regression model was chosen from a stepwise selection based on the Akaike Information Criterion (AIC). The analyses were performed on Prism 6 or SAS 9.4 software packages and all significance levels were fixed at 0.05.All data were tested for normality using the Shapiro-Wilk test and according to the distribution, parametric or non-parametric tests were applied. Differences between the groups were assessed by one-way ANOVA or Kruskal-Wallis test. Tukey or Dunn’s tests were used as In this study 88 patients with a positive nasopharyngeal throat swab for COVID-19 were included: 11 non-hospitalized (NH), 37 hospitalized in a conventional care unit (H) and 40 hospitalized in intensive care unit (ICU). Blood samples were collected at the admission D1, D5 and D14. Symptoms started on average 9 days before admission to the emergency department. Consequently, samples obtained at D1, D5 and D14 correspond to day 9, 14 and day 23 after initial symptoms. 21 healthy subjects (HC) ) were used as controls.Baseline characteristics of controls and patients are reported in Routine biological analyses of the COVID-19 patients are reported in +CD16-) (p<0.01) and non-classical (CD14-CD16+) monocytes (p<0.0001) (+) (p<0.0001) and both subtypes of NK cells (NK CD56bright and NK CD56dim) (p<0.0001) than HC (+CD141-) as compared to HC (+CD16+) was observed in the H group compared to HC (p<0.05), while no significant differences were observed for mDCs type 1 ; COVID-19 D5: 130.3 ng/mL (115.7-175.4); COVID-19 D14: 149.4 ng/mL (135.5-186.4) versus HC: 75.9 ng/mL (63.7-94.3), median of all patients (95% CI), p<0.0001). When comparing each subgroup of COVID-19 patients with HC, plasma chemerin levels were increased in the ICU group at all timepoints , in the H group at D1 and D14 and in the NH group at D1  and at all time points, for each increase of 1 mg/dL of CRP, chemerin increased of 209.2 pg/mL , p<0.05) Figure 3As expected, the duration of hospitalization was lower in the H group compared to the ICU group –10 days Regarding mortality, 16 out of 88 (18%) COVID-19 patients died, 1 from the H group (2.7%) and 15 from the ICU group (37.5%). Chemerin levels were higher in patients who deceased at all time points Table 3.Receiver operating characteristic (ROC) curves were also generated to evaluate the predictive value of plasma chemerin on mortality. This showed that day 14 chemerin’s concentration best estimated mortality risk  compared to D1  and D5 concentrations   protein, a molecule that is predominantly expressed by damaged alveolar type II cells . This anSince we observed higher levels of chemerin in the blood of COVID-19 patients we determined the expression of its main receptor ChemR23 at the surface of immune cell populations of patients and HC. Small increases in ChemR23 expression were found on the classical and intermediate monocyte and mDC populations when comparing some categories of COVID-19 patients and HC , no exprFinally, we determined the expression of chemerin and ChemR23 in lung tissues from autopsied COVID-19 patients. Given the important heterogeneity of lung lesions, we selected areas with typical diffuse alveolar damage (DAD) or pneumonia/bronchopneumonia. Areas with DAD were either in exudative phase with hyaline membranes, interstitial and intra-alveolar edema, microthrombi or in organizing phase with interstitial or intra-alveolar proliferation of myofibroblasts and proliferation of type II pneumocytes . Lysis pChemR23 expression was found on smooth muscle cells of vessels and bronchi  are in line with those observed in rheumatoid arthritis (154 ng/mL ), psoriaImportantly, chemerin concentrations at day 14 was associated with risk of mortality in both univariate logistic regression and multivariate analysis. Moreover, we identified a chemerin threshold of 291.4 ng/mL at day 23 after initial symptoms that could predict mortality with a high sensitivity, high specificity and an interesting high negative predictive value. In our cohort, patients with hypertension had higher chemerin levels. This is in agreement with a previously study demonstrating that chemerin was independently associated with hypertension .The increased levels of chemerin detected in plasma of COVID-19 patients were not observed in BAL and they were much lower than the ones observed in blood. This could be explained by the important dilution factor of the BAL and possible degradation of chemerin by proteases. Also, no BAL were obtained from healthy subjects and the number of BAL collected was relatively small and from heterogenous patients . All of these could interfere with our results and further studies are therefore necessary to determine chemerin levels in the BAL of these patients. Of note, we were unable to find studies where chemerin levels were measured in BAL for comparison.We previously showed that ChemR23 KO mice exhibited a similar immune signature as severe forms of COVID-19 with development of a cytokine storm and lower levels of pDCs and lymphocytes . We confvia activation of the Akt/eNOS/NO pathway  of both HC and COVID-19 patients. Interestingly, endothelial activation and dysfunction are involved in the physiopathology of severe viral pneumonia and seems to be more prominent in cases of severe SARS-CoV-2 infection. Indeed, it has been demonstrated that patients who died from COVID-19 had predominant angiocentric inflammation and more severe endothelial injury than patients with influenza pneumonia of equal severity . It has  pathway . Additio pathway , 44. TheBesides ECs we observed that chemerin is expressed in fibroblasts/myofibroblasts in lesions of late DAD and ChemR23 is also expressed on smooth muscle cells. Recent studies demonstrated that epithelial-to-mesenchymal and endothelial-to-mesenchymal transition were present in patients with severe COVID-19 , 46 and Our study has few limitations. It is a monocentric study with a relatively small number of patients, particularly in the HC and NH groups, that may decrease the robustness of our results. Nevertheless, our patient cohort is quite representative of hospitalized COVID-19 patients with a predominance of elderly men with numerous comorbidities such as hypertension and diabetes . As wellIn conclusion, our study demonstrates that increased plasma chemerin levels are a marker of severity and death in COVID-19 patients. However, multicentric studies and validation cohorts are needed to affirm chemerin as a biomarker of severity and death to be used in daily clinical practice. Further studies are also needed to identify the precise mechanisms by which the chemerin/ChemR23 system affects ARDS secondary to viral pneumonia and its possible role in lung fibrosis.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Local Ethical Committee, (P2020/238 and P2020/232), Erasme Hospital. The patients/participants provided their written informed consent to participate in this study.The protocol was designed by PL, VC, MP, AK, and BB. The clinical data collection was performed by PL, SM, and CO. Technical support and material were provided by VC, VO, ND, VDM, AM, CD, FM, PB, and IS. Experiments were realized by PL, NA and NDV. Data analysis was performed by PL, NA, VC, NDV, MR, MP, AK, and BB. The manuscript was written by PL, AK, and BB. All authors critically reviewed the manuscript and approved its final version.PL has a PhD scholarship from F.N.R.S.  and is a grant holder of Fonds Erasme. NA was supported partially by a ULB grant and partially from Fonds Erasme. VC was supported by a COVID-19 ULB grant. ND is a post-doctoral clinical master specialist of the F.N.R.S. V.d.M. received a grant from the Foundation Jaumotte-Demoulin. CD is a Senior Research Associate with the F.N.R.S. The CMMI is supported by the European Regional Development Fund and the Walloon Region . The department of Pathology of Erasme hospital  was supported by Fonds Yvonne Boël, Foundation ULB and Fonds Erasme. BB and FM received grants from Foundation ULB and Fonds Erasme. NDV obtained a LHUB-ULB research grant. BB received financial support from Amgen and Boehringer Ingelheim. This article was published with the financial support of the Fonds Erasme.We would like to thank Audrey Godefroid, Anne Van Praet and Noémie Law-Weng-Sam (LoVMI) for blood samples handling, biobanking and Multiplex analysis; Flavienne Sandras  for bronchoalveolar lavages and tissues biobanking; Justine Allard and Amandine Collin  for technical assistance on immunohistochemistry; Mélina Houinsou Hans  for help with statistical analysis.BB received a financial support from Amgen and Boehringer Ingelheim. These funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article and the decision to submit for publication.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.